PROJECT SUMMARY: Oct4 is a master transcriptional regulator of pluripotency. Intriguingly, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) co-express Oct4 together with two paralogs, Oct1 and Oct6. These proteins bind the same sequences in vitro and many of the same genes in vivo. Their functions in pre- implantation embryos and ESCs, and immediately following differentiation, are unknown. Though critical early developmental fate decisions are made as ESCs first lose pluripotency and Oct4 is lost, how this process works at a molecular level remains a black box. For example, developmentally poised Oct4 targets can be induced many days after loss of Oct4 itself, raising the question of how poising is maintained. This proposal focuses on Oct1/Oct6/Oct4 collaboration in pluripotent cells and early during differentiation. We previously showed that Oct1 either represses gene transcription, by associating with NuRD, or maintains silent genes in a configuration that allows for later expression, by associating with Jmjd1a/KDM3A to decrease local histone H3K9 methylation. More recently we showed that Oct1 is dispensable in undifferentiated ESCs, but following differentiation is required both to induce developmental-specific genes, and to suppress genes specific for alternative developmental lineages. As a consequence, Oct1 deficient ESCs appear normal but show severe defects upon differentiation. Oct1 does not occupy these targets in ESCs, presumably due to competition from the more abundant Oct4 protein. Instead, Oct1 occupies these genes as ESCs differentiate and Oct4 is lost. We propose a ?handoff? model whereby silent but poised genes are occupied by Oct1, and possibly Oct6, to maintain proper developmental gene expression. We will test this model by identifying common and unique Oct1/Oct4/Oct6 target sites genome-wide in differentiating ESCs, determining whether handoff of cofactors accompanies Oct4 replacement by Oct1 in differentiating cells, and studying Oct1-mediated repression of lineage-inappropriate genes, which are ectopically expressed upon differentiation of Oct1 deficient ESCs. Aim 1: Test the validity of the handoff model. Aim 2: Define the cofactors used by Oct1 and Oct4 at developmentally inducible targets in pluripotent cells and immediately after differentiation. Aim 3: Determine the mechanism by which Oct1 represses lineage-inappropriate genes.